Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
  • A61K31/575—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
  • A61K47/6949—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit inclusion complexes, e.g. clathrates, cavitates or fullerenes
  • A61K47/6951—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit inclusion complexes, e.g. clathrates, cavitates or fullerenes using cyclodextrin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0048—Eye, e.g. artificial tears
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P27/00—Drugs for disorders of the senses
  • A61P27/02—Ophthalmic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P27/00—Drugs for disorders of the senses
  • A61P27/02—Ophthalmic agents
  • A61P27/12—Ophthalmic agents for cataracts
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B82—NANOTECHNOLOGY
  • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
  • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07J—STEROIDS
  • C07J9/00—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/64—Fluorescence; Phosphorescence
  • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2201/00—Features of devices classified in G01N21/00
  • G01N2201/10—Scanning
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
  • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
  • G01N2333/4701—Details
  • G01N2333/4709—Amyloid plaque core protein
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2500/00—Screening for compounds of potential therapeutic value
  • G01N2500/20—Screening for compounds of potential therapeutic value cell-free systems

Definitions

• the disclosure generally relates to inhibitors of ⁇ -crystallin aggregation, uses thereof, and methods of screening for therapeutically effective modulators of protein aggregation.
• Cataract or a clouding of the eye lens
• Cataract is a condition affecting over half of all adults over the age of 80, with approximately 25 million patients suffering from the condition in the United States.
• cataracts are thought to be the leading cause of blindness worldwide.
• ⁇ A-crystallin (cryAA) and ⁇ B-crystallin (cryAB) comprise thirty percent of the protein content of the eye lens, where they are responsible for maintaining lens transparency ( Haslbeck et al., Nat Struct Mol Biol 12, 842 (2005 ).
• cryAA and cryAB belong to a family of small heat shock proteins (sHSPs) that contain a conserved crystallin domain ( Bloemendal et al., Prog Biophys Mol Biol 86, 407 (2004 ); Haslbeck, supra ). Once synthesized, these lens sHSPs are never degraded, so any damage accumulates through life and eventually leads to aging-associated cataract (Haslbeck, supra ; Perng et al., J Biol Chem 274, 33235 (1999 ); Meehan et al., J Biol Chem 279, 3413 (2004 ); Meehan et al., J Mol Biol 372, 470 (2007 )).
• sHSPs small heat shock proteins
• cryAB destabilizing mutations in cryAB, such as R120G, result in hereditary forms of cataract with early onset ( Vicart et al., Nat Genet 20, 92 (1998 )).
• hereditary cataract cryAB is prone to aggregation and forms amyloid-like fibrils in vitro ( Andley et al., PLoS One 6, e17671 (2011 )).
• the invention provides a method of treating or preventing cataract, the method comprising administering to an individual in need thereof an effective amount of a composition comprising a compound of formula I: wherein:
• the invention also provides an ophthalmic pharmaceutical composition
• a pharmaceutically acceptable ophthalmic carrier and a compound of formula I.
• the compound of formula I has a structure of formula IA or formula IB: wherein each R 11 is independently alkyl, CO 2 H, or CO 2 alkyl.
• the compound has a structure of formula II: wherein R 12 is H or OH and R 13 is H or OH.
• the compound is 5-cholestin-3b,25-diol.
• the composition is administered topically, subconjunctivally, retrobulbarly, periocularly, subretinally, suprachoroidally, or intraocularly.
• the cataract is an age-related cataract or a diabetic cataract.
• the individual has a hereditary form of cataract with early onset.
• the individual has a R120G mutation and/or a D109H mutation in cryAB.
• the pharmaceutically acceptable ophthalmic carrier is a cyclodextrin.
• the cyclodextrin is (2-hydroxypropyl)- ⁇ -cyclodextrin.
• the invention includes a high-throughput method of screening compounds for modulation of protein thermal stability, the method comprising: (a) contacting a protein with each of a plurality of test compounds; and (b) measuring the melting transition (T m ) of the protein in the presence of each of the plurality of test compounds, wherein a compound that decreases or increases the apparent T m by at least 2 standard deviations is a pharmacological protein chaperone.
• the protein is an amyloid-forming protein or a protein underlying a loss-of-function disease.
• the amyloid-forming protein is selected from the group consisting of Hsp27, ⁇ A-crystallin, ⁇ B-crystallin, ⁇ B2-crystallin, ⁇ B1-crystallin, yD-crystallin, Hsp22, Hsp20, tau, Alpha-synuclein, IAPP, beta-amyloid, PrP, Huntingtin, Calcitonin, Atrial natriuretic factor, Apolipoprotein AI, Serum amyloid A, Medin, Prolactin, Transthyretin, Lysozyme, Beta 2 microglobulin, Gelsolin, Keratoepithelin, Cystatin, Immunoglobulin light chain AL, and S-IBM.
• the protein underlying a loss-of-function disease is selected from the group consisting of mutant ⁇ -glucosidase, cystic fibrosis transmembrane receptor, hexosaminidase A, hexosaminidase B, ⁇ -galactosidase, and alpha-glucosidase.
• the T m is determined using a high-throughput differential scanning fluorimetry device.
• the measuring step comprises: (b1) heating the protein in the presence of each of a plurality of test compounds from 50°C to 80°C, (b2) cooling the protein to 25°C, (b3) maintaining the protein at 25°C for 10 seconds, and (b4) measuring the fluorescence of the protein.
• the method further comprises repeating steps (bl)-(b4) between 2 and 30 times, wherein each repeat of step (b1) is performed at an incrementally higher temperature.
• the amyloid-forming protein is heated from 65°C to 80°C in 1°C increments.
• (b1) further comprises, after heating, equilibrating the amyloid-forming protein and test compound between 60 and 180 seconds. In various aspects of the method, the equilibrating step is 130 seconds.
• the invention includes a high-throughput screening system, comprising: (a) an amyloid-forming protein; (b) a device capable of measuring the melting transition (T m ) of the amyloid-forming protein; and (c) a plurality of test compounds.
• the protein is selected from the group consisting of Hsp27, ⁇ A-crystallin, ⁇ B-crystallin, ⁇ B2-crystallin, ⁇ B1-crystallin, ⁇ D-crystallin, Hsp22, Hsp20, tau, Alpha-synuclein, IAPP, beta-amyloid, PrP, Huntingtin, Calcitonin, Atrial natriuretic factor, Apolipoprotein AI, Serum amyloid A, Medin, Prolactin, Transthyretin, Lysozyme, Beta 2 microglobulin, Gelsolin, Keratoepithelin, Cystatin, Immunoglobulin light chain AL, and S-IBM.
• the device is a high-throughput differential scanning fluorimetry device.
• the use of the compound of any one of structural formulas I, IA, IB, or II in any of the methods disclosed herein or for preparation of medicaments for administration according to any of the methods disclosed herein is specifically contemplated.
• the invention provides a compound of any one of structural formulas I, IA, IB, or II for use in a method of treating or preventing cataract, wherein the method comprises administering to an individual in need thereof an effective amount the compound.
• the invention provides inhibitors of ⁇ -crystallin aggregation and methods of using ⁇ -crystallin aggregation inhibitors to, e.g., treat or prevent cataracts in a subject having or at risk of developing cataracts.
• Inhibitors of ⁇ -crystallin aggregation of the invention are, e.g, sterols represented by Formula I, Formula IA, Formula IB, and Formula II and may be formulated in ophthalmic pharmaceutical compositions comprising a pharmaceutically acceptable ophthalmic carrier. Because cataracts affect such a large portion of the population (over half of all adults over the age of 80) and the predominant treatment is surgical intervention, the discoveries described herein represent a significant advancement in the non-surgical methodologies available for treating cataracts.
• compounds of the invention are able to reverse aggregation of ⁇ -crystallin and inhibit further aggregation of ⁇ -crystallin.
• the invention further provides high-throughput methods of screening compounds for modulation of protein thermal stability, the method comprising contacting a protein with each of a plurality of test compounds; and measuring the melting transition (T m ) of the protein in the presence of each of the plurality of test compounds, wherein a compound that decreases or increases the apparent T m by at least 2 standard deviations is identified as a pharmacological protein chaperone.
• the invention provides a method of treating or preventing cataract, the method comprising administering to an individual in need thereof an effective amount of a composition comprising a compound of any one of structural formulae I, IA, IB, or II, or e.g., a compound in Table 1.
• the cataract is an age-related cataract, a diabetic cataract, a cataract associated with surgery, a cataract resulting from exposure to radiation, a cataract resulting from a genetic illness, a cataract resulting from an infection, or a cataract resulting from medication.
• the individual has a hereditary form of cataract with early onset.
• hereditary forms of cataract include individuals with a R120G mutation and/or a D109H mutation in cryAB.
• An individual "in need of” treatment according to the invention is an individual that is suffering from a cataract.
• the individual may have an age-related cataract or a cataract resulting from having diabetes.
• an individual "in need of” treatment according to the invention is an individual that is at risk for developing a cataract.
• Individuals at risk of developing a cataract include, but are not limited to, individuals with a family history of developing cataracts, individuals with a mutation linked to a cataract with early onset, such as individuals with a R120G mutation and/or a D109H mutation in cryAB, individuals exposed to radiation, diabetics, and the like.
• the individual has been diagnosed with cataract in one eye, and the compound is administered to prevent or slow cataract formation in the contralateral eye.
• the invention reduces the cataract, e.g., at least about 5%, at least about 10% or at least about 20% compared to levels observed in the absence of the inventive method (e.g., in a biologically-matched control subject or specimen that is not exposed to the compound of the inventive method).
• the cataract is reduced by at least about 30%, at least about 40%, at least about 50%, or at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more (about 100%).
• the "treating" cataracts according to inventive method inhibits cataract formation by, e.g., at least about 5%, at least about 10% or at least about 20% compared to levels observed in the absence of the inventive method (e.g., in a biologically-matched control subject or specimen that is not exposed to the compound of the inventive method).
• cataract formation is inhibited by at least about 30%, at least about 40%, at least about 50%, or at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more (about 100%) compared to cataract formation in the absence of the compound of the inventive method.
• Cataracts generally are detected using any of a number of optic tests including, but not limited to, visual acuity testing, ophthalmoscopy, slit-lamp examination, keratometry, tonometry, contrast testing, glare sensitivity, wavefront mapping.
• an "effective amount" of a composition comprising a compound of any one of structural formulae I, IA, IB, or II, or a compound in Table 1 is an amount that inhibits or reduces aggregation of an amyloid-forming protein such as cryAB in an individual. Inhibiting aggregation does not require a 100% inhibition of aggregation. Any inhibition of aggregation constitutes a beneficial biological effect in a subject. In this regard, the invention inhibits the aggregation of an amyloid-forming protein, e.g., at least about 5%, at least about 10% or at least about 20% compared to levels observed in the absence of the inventive method (e.g., in a biologically-matched control subject or specimen that is not exposed to the compound of the inventive method).
• the formation of amyloid aggregates is inhibited by at least about 30%, at least about 40%, at least about 50%, or at least about 60%.
• the inventive method inhibits amyloid formation by at least about 70%, at least about 80%, at least about 90%, or more (about 100%) compared to amyloid formation in the absence of the compound of the inventive method.
• an "effective amount" for reducing aggregation does not require a 100% abolition of aggregation.
• Any reduction of aggregation constitutes a beneficial biological effect in a subject.
• the invention reduces the aggregation of an amyloid-forming protein, e.g., at least about 5%, at least about 10% or at least about 20% compared to levels observed in the absence of the inventive method (e.g., in a biologically-matched control subject or specimen that is not exposed to the compound of the inventive method).
• amyloid aggregates are reduced by at least about 30%, at least about 40%, at least about 50%, or at least about 60%.
• the inventive method reduces amyloid aggregates by at least about 70%, at least about 80%, at least about 90%, or more (about 100%) compared to amyloid aggregates in the absence of the compound of the inventive method.
• the most appropriate method of administering a compound to a subject is dependent on a number of factors.
• the compound according to the invention is administered locally to the eye, e.g., topically, subconjunctivally, retrobulbarly, periocularly, subretinally, suprachoroidally, or intraocularly.
• the composition is delivered locally to the eye via injection. Injectable solutions can be directly injected into the cornea, crystalline lens and vitreous or their adjacent tissues using a fine needle. The composition also can be administered as an intraocular perfusate.
• Additional contemplated routes of administration include, but are not limited to, one or more of: oral (e.g., as a tablet, capsule, or as an ingestible solution), mucosal (e.g., as a nasal spray or aerosol for inhalation), nasal, parenteral (e.g., by an injectable form), gastrointestinal, intraspinal, intraperitoneal, intramuscular, intravenous, intrauterine, intradermal, intracranial, intratracheal, intravaginal, intracerebroventricular, intracerebral, subcutaneous, transdermal, rectal, buccal, epidural and sublingual.
• oral e.g., as a tablet, capsule, or as an ingestible solution
• mucosal e.g., as a nasal spray or aerosol for inhalation
• nasal parenteral (e.g., by an injectable form)
• gastrointestinal e.g., intraspinal, intraperitoneal, intramuscular, intravenous, intrauter
• the mode for delivery of a composition of the invention to the eye is via a contact lens.
• the lens may be provided pre-treated with the desired compound.
• the lens is provided in a kit with components for preparing a coated lens, which are provided as lyophilized powders for reconstitution or as concentrated or ready-to-use solutions.
• the compositions can be provided as kits for single or multi-use.
• the mode for delivery of a composition of the invention to the eye is via an ophthalmic rod ( Gwon et al., Ophthalmology. 1986 Sep;93(9 Suppl):82-5 ). In some embodiments, the mode for delivery of a composition of the invention to the eye is via an intraocular lens-hydrogel assembly ( Garty et al., Invest Ophthalmol Vis Sci, 2011 Aug 3;52(9):6109-16 ).
• the composition comprising the compound is provided in a therapeutically effective amount that achieves a desired biological effect at a medically-acceptable level of toxicity.
• the dosage of the compositions may vary depending on the route of administration and the severity of the disease. The dosage may also be adjusted depending on the body weight, age, sex, and/or degree of symptoms of each patient to be treated. The precise dose and route of administration will ultimately be at the discretion of the attendant physician or veterinarian. It will be appreciated that it may be necessary to make routine variations to the dosage depending on the age and weight of the patient as well as the severity of the condition to be treated.
• the frequency of administration depends on the formulation and the aforementioned parameters. For example, it may be desirable to apply eye drops at least once per day, including 2, 3, 4, or 5 times per day.
• Exemplary doses of the compounds for administration to a human (of approximately 70 kg body weight) via an ocular route are 0.1 mg to 1 g, e.g., 1 mg to 500 mg of the compound per unit dose. In various embodiments, the dose is about 1 ⁇ g/kg body weight to 15 mg/kg body weight.
• the dose can be 1 ⁇ g/kg, 5 ⁇ g/kg, 10 ⁇ g/kg, 20 ⁇ g/kg, 30 ⁇ g/kg, 40 ⁇ g/kg, 50 ⁇ g/kg, 60 ⁇ g/kg, 70 ⁇ g/kg, 80 ⁇ g/kg, 90 ⁇ g/kg, 100 ⁇ g/kg, 120 ⁇ g/kg, 140 ⁇ g/kg, 160 ⁇ g/kg, 180 ⁇ g/kg, 200 ⁇ g/kg, 300 ⁇ g/kg, 400 ⁇ g/kg, 500 ⁇ g/kg, 600 ⁇ g/kg, 700 ⁇ g/kg, 800 ⁇ g/kg, 900 ⁇ g/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, 11 mg/kg, 12 mg/kg, 13 mg/kg, 14 mg/kg, or 15 mg/kg.
• Preferred concentrations of the compound of formula I, IA, IB, or II, or a compound listed in Table 1, range from about 1 ⁇ g/ml to 500 ⁇ g/ml, for example, about 1 ⁇ g/ml, about 2 ⁇ g/ml, about 3 ⁇ g/ml, about 4 ⁇ g/ml, about 5 ⁇ g/ml, about 10 ⁇ g/ml, about 20 ⁇ g/ml, about 30 ⁇ g/ml, about 40 ⁇ g/ml, about 50 ⁇ g/ml, about 60 ⁇ g/ml, about 70 ⁇ g/ml, about 80 ⁇ g/ml, about 90 ⁇ g/ml, about 100 ⁇ g/ml, about 120 ⁇ g/ml, about 140 ⁇ g/ml, about 160 ⁇ g/ml, about 180 ⁇ g/ml, about 200 ⁇ g/ml, about 250 ⁇ g/ml, about 300 ⁇ g/ml, about 350 ⁇ g/
• composition according to the invention is provided in a container, either as a concentrate that is diluted prior to use in an appropriate diluent or at the ready-to-use concentration.
• a container either as a concentrate that is diluted prior to use in an appropriate diluent or at the ready-to-use concentration.
• single dosages are provided in sterile vials.
• the compound of the inventive method or composition is a compound of formula I: wherein:
• the compound of the inventive method or composition is a compound of formula IA or formula IB: wherein each R 11 is independently alkyl, CO 2 H, or CO 2 alkyl.
• the compound has a structure of formula II: wherein R 12 is H or OH and R 13 is H or OH. In some embodiments, the compound is 5-cholestin-3b,25-diol.
• the compound is a compound listed in Table 1.
• the invention further comprises a composition comprising the compound of any one of structural formulae I, IA, IB, or II, or a compound listed in Table 1, and a pharmaceutically acceptable ophthalmic carrier, e.g., a pharmaceutically acceptable excipient, carrier, binder, and/or diluent.
• a pharmaceutically acceptable ophthalmic carrier e.g., a pharmaceutically acceptable excipient, carrier, binder, and/or diluent.
• the composition includes a free acid, free base, salt (e.g., an acid or base addition salt), hydrate or prodrug of the compound of any one of structural formula I, IA, IB, or II, or a compound listed in Table 1.
• the prodrug is a material that includes the compound in any one of structural formulae I, IA, IB, or II, or a compound listed in Table 1, covalently bound to a carrier moiety.
• the carrier moiety can be released from the compound in any one of structural formulae I, IA, IB, or II, or a compound listed in Table 1, in vitro or in vivo to yield compound in any one of structural formula I, IA, IB, or II, or a compound listed in Table 1.
• Prodrug forms are well known in the art as exemplified in Sloan, K. B., Prodrugs, M. Dekker, New York, 1992 ; and Testa, B. and Mayer, J. M., Hydrolysis in drug and prodrug metabolism: chemistry, biochemistry, and enzymology, Wiley-VCH, Zurich, 2003 .
• the composition comprises the compound of any one of structural formulae I, IA, IB, or II, or a compound listed in Table 1, formulated as eye drops, injectable solutions or eye ointments.
• These pharmaceutical compositions can be formulated by admixing, diluting or dissolving the compound, optionally, with appropriate pharmaceutical additives such as excipients, disintegrators, binders, lubricants, diluents, buffers, antiseptics, moistening agents, emulsifiers, dispersing agents, stabilizing agents and dissolving aids in accordance with conventional methods and formulating in a conventional manner depending upon the dosage form.
• eye drops can be formulated by dissolving the compound in sterilized water in which a surface active agent is dissolved and optionally adding appropriate pharmaceutical additives such as a preservative, a stabilizing agent, a buffer, an antioxidant and a viscosity improver.
• the composition includes a cyclodextrin.
• the cyclodextrin is (2-hydroxypropyl)- ⁇ -cyclodextrin.
• Physiologically acceptable buffers include, but are not limited to, a phosphate buffer or a Tris-HCl buffer (comprising tris(hydroxymethyl)aminomethane and HCl).
• a Tris-HCl buffer having pH of 7.4 comprises 3 g/l of tris(hydroxymethyl)aminomethane and 0.76 g/l of HCl.
• the buffer is lOx phosphate buffer saline (“PBS”) or 5x PBS solution.
• buffers include, but are not limited to, buffers based on HEPES (N- ⁇ 2-hydroxyethyl ⁇ peperazine-N'- ⁇ 2-ethanesulfonic acid ⁇ ) having pK a of 7.5 at 25°C and pH in the range of about 6.8-8.2; BES (N,N-bis ⁇ 2-hydroxyethyl ⁇ 2-aminoethanesulfonic acid) having pK a of 7.1 at 25°C and pH in the range of about 6.4-7.8; MOPS (3- ⁇ N-morpholino ⁇ propanesulfonic acid) having pK a of 7.2 at 25°C and pH in the range of about 6.5-7.9; TES (N-tris ⁇ hydroxymethyl ⁇ -methyl-2-aminoethanesulfonic acid) having pK a of 7.4 at 25°C and pH in the range of about 6.8-8.2; MOBS (4- ⁇ N-morpholino ⁇ butanesulfonic acid) having pK a of
• polymers e.g., cellulose derivatives such as hydroxypropylmethylcellulose (HPMC) and hydroxypropylcellulose (HPC), poly(acrylic acid) (PAA), polyacrylates, cyclodextrins and natural gums, polyorthoesters (POEs) and mucoadhesive polymers
• semisolids such as gels, films and other inserts
• resins such as ion exchange resins; iontophoretic delivery
• colloidal particles such as microspheres and nanoparticles
• the compounds of the invention may also be provided in combination with other therapeutic agents.
• the compounds of the invention may be co-formulated with a pain reliever, an anesthetic, artificial tears, an enzyme inhibitor, a cytokine inhibitor, an anti-inflammatory, or an antibiotic
• the antibiotic is an antibacterial, antiviral, antifungal, antiprotozoal agent, or a combination thereof.
• the compounds of the invention may also be provided in combination with an ocular therapeutic selected from the group consisting of Acular (ketorolac tromethamine ophthalmic solution) 0.5%, Acuvail (ketorolac tromethamine), AK-Con-A (naphazoline ophthalmic), Akten (lidocaine hydrochloride), Alamast, Alphagan (brimonidine), Alrex, Astepro (azelastine hydrochloride nasal spray), AzaSite (azithromycin), Bepreve (bepotastine besilate ophthalmic solution), Besivance (besifloxacin ophthalmic suspension), Betaxon, BSS Sterile Irrigating Solution, Cosopt, Durezol (difluprednate), Eylea (aflibercept), Lotemax, Lucentis (ranibizumab), Lumigan (bimatoprost ophthalmic solution), Macugen (pegaptanib), Ocuflox (of
• the invention includes a high-throughput method of screening compounds for modulation of protein thermal stability.
• the method comprises (a) contacting a protein with each of a plurality of test compounds; and (b) measuring the melting transition (T m ) of the protein in the presence of each of the plurality of test compounds, wherein a compound that decreases or increases the apparent T m by at least 2 standard deviations is a pharmacological protein chaperone.
• a compound that decreases or increases the apparent T m by at least 3 standard deviations is a pharmacological protein chaperone.
• DSF differential scanning fluorimetry
• T m the melting transition of the protein target is measured in the presence of potential ligand.
• DSF measures thermal unfolding of the target protein via the fluorescence of intrinsic tryptophans, or a dye, such as 1,8-anilinonapthalenesulfonic (bis-ANS) or Sypro Orange. Binding of a ligand adds free energy to the folded state or key intermediates, which limits unfolding and shifts the apparent T m .
• the melting transition is determined using a high-throughput differential scanning fluorimetry device, such as a ThermoFluor® 384-well DSF platform, or a real-time PCT thermocycler.
• the step wherein the melting transition is measured comprises the following steps: (b1) heating the protein in the presence of each of a plurality of test compounds from 50°C to 80°C, (b2) cooling the protein to 25°C, (b3) maintaining the protein at 25°C for 10 seconds, and (b4) measuring the fluorescence of the protein.
• the method further comprises repeating steps (bl)-(b4) between 2 and 30 times, wherein each repeat of step (b1) is performed at an incrementally higher temperature. For example, during the first iteration, the protein is heated to 65°C in the presence of a test compound and cooled to 25°C where it is maintained for about 10 seconds before the fluorescence of the protein is measured.
• the protein is heated to 66°C in the presence of a test compound and cooled to 25°C where it is maintained for about 10 seconds before the fluorescence of the protein is measured. This process is repeated (e.g., between 2 and 30 times) while increasing the peak temperature to which the protein is heated, e.g., in 1°C increments.
• the protein is equilibrated at the peak temperature between 60 and 180 seconds. In specific embodiments of the method, the equilibrating step is 130 seconds.
• the step wherein the melting transition is measured comprises gradually heating the protein in the presence of each of a plurality of test compounds to 80°C while continuously measuring the fluorescence of the protein.
• the protein is an amyloid-forming protein.
• the desired modulator decreases the melting transition of the protein.
• Such a modulator stabilizes the non-amyloid form of the protein.
• the amyloid-forming protein is selected from the group consisting of Hsp27, ⁇ A-crystallin (cataract), ⁇ B-crystallin (cataract), ⁇ B2-crystallin (cataract), ⁇ B1-crystallin (cataract), ⁇ D-crystallin (cataract), Hsp22, Hsp20, tau, Alpha-synuclein (Parkinson's disease), IAPP (Diabetes mellitus type 2), beta-amyloid (Alzheimer's disease), PrP (Transmissible spongiform encephalopathy), Huntingtin (Huntington's Disease), Calcitonin (Medullary carcinoma of the thyroid), Atrial natriuretic factor (Isolated atrial amyloidosis), Apolipoprotein AI (Atherosclerosis), Serum amyloid A (Rheumatoid arthritis), Medin (Aortic medial amyloid), Prol
• the protein is a protein underlying a loss-of-function disease.
• the desired modulator increases the melting transition of the protein.
• Such a modulator stabilizes the mutant form of the protein such that it remains active (i.e., degradation of the protein is reduced).
• the protein underlying a loss-of-function disease can be, for example, mutant ⁇ -glucosidase, mutant glucosylceramidase (Gaucher's disease), mutant cystic fibrosis transmembrane receptor (cystic fibrosis), mutant hexosaminidase A (Tay-Sachs disease), mutant hexosaminidase B (Sandhoff disease), mutant ⁇ -galactosidase (Morquio syndrome), and mutant alpha-glucosidase (i.e., Pompe disease).
• the invention includes a high-throughput screening system, comprising: (a) an amyloid-forming protein; (b) a device capable of measuring the melting transition (T m ) of the amyloid-forming protein; and (c) a plurality of test compounds.
• the device capable of measuring the T m of the amyloid-forming protein can be any device known in the art.
• the device is a differential scanning fluorimetry device, such as a ThermoFluor® 384-well DSF platform.
• the protein is selected from the group consisting of Hsp27, ⁇ A-crystallin, ⁇ B-crystallin, ⁇ B2-crystallin, ⁇ B1-crystallin, ⁇ D-crystallin, Hsp22, Hsp20, tau, Alpha-synuclein, IAPP, beta-amyloid, PrP, Huntingtin, Calcitonin, Atrial natriuretic factor, Apolipoprotein AI, Serum amyloid A, Medin, Prolactin, Transthyretin, Lysozyme, Beta 2 microglobulin, Gelsolin, Keratoepithelin, Cystatin, Immunoglobulin light chain AL, and S-IBM.
• This Example describes a high-throughput method of screening for compounds that stabilize an exemplary aggregation-prone protein, Hsp27.
• DSF Differential scanning fluorimetry
• Hsp27 was used for the HTS because it readily forms amyloids and it retains the highly conserved crystallin domain found in all sHSPs. Hsp27 was soluble at room temperature, but it thermally unfolded and rapidly formed stable amyloids upon heating.
• the screen was performed in 7 ⁇ L volume, with 10 ⁇ M Hsp27 in 50 mM NaPO4, pH 7.4, 700 mM NaCl, 50 mM LiCl, and 100 ⁇ M bis-ANS in Abgene black 384-well PCR plates. Reactions were covered with silicon oil to limit evaporation. Control tests suggested that the assay tolerated up to 4% DMSO but 1% was used as a final concentration. The plates were measured in up/down mode, which was empirically determined to give better signal-to-noise than the continuous ramp mode.
• Plates were heated from 65°C to 80°C in 1°C increments, equilibrated for 130 seconds at each high temperature, cooled to 25°C, and held for 10 seconds at 25°C prior to imaging with a single 10 second exposure for each temperature reading. Plate uniformity tests measured the T m of Hsp27 at 72.3 ⁇ 0.16 °C. The Z factor was calculated to vary between ⁇ 0.59 and 0.71 and the CVs were 8%. Most of the variability appeared to arise from some "edge effects"
• these studies validate the HTS method as a robust means for identifying pharmacological chaperones for amyloid-forming proteins, such as cryAB. Indeed, the studies validate the HTS method for finding agents that modulate T m for any protein wherein increased (e.g., loss of function proteins such as the mutant form of alpha-glucosidase responsible for Pompe Disease) or decreased stability (aggregation-prone proteins such as Huntingtin) is desired.
• increased e.g., loss of function proteins such as the mutant form of alpha-glucosidase responsible for Pompe Disease
• decreased stability aggregation-prone proteins such as Huntingtin
• This Example describes structure-activity relationship (SAR) studies of a sterol scaffold identified in the primary screen of Example 1. As described further below, the SAR revealed a general chemical structure (Formula I) for compounds that reduced the T m of an amyloid-forming protein, R120G cryAB, by at least 2°C.
• Biolayer interferometry allows for the identification of false positives.
• biotinylated cryAB is immobilized to streptavidin-coated pins and equilibrium association data across a hundred-fold concentration range is analyzed to generate the apparent K D .
• the response of a biocytin-blocked pin is subtracted from each sensorgram.
• Example 2 demonstrates that the compounds identified in Example 1 suppress amyloid formation. Significantly, the compounds are also able to reverse amyloid formation.
• 5-cholesten-3b,25-diol To explore the mechanism of action of 5-cholesten-3b,25-diol, the binding site on R120G cryAB was explored by 15 N HSQC NMR, and analysis of the resulting chemical shifts suggested that it binds an exposed face of the conserved crystallin domain. Specifically, 5-cholesten-3b,25-diol binds a groove across the cryAB dimer interface and makes contacts with residues near R120 and D109.
• the R120G cryAB knock-in mouse is a clinically-acceptable model of aging-associated and hereditary cataract.
• the animal model develops severe cataracts within 20 weeks ( Andley et al., PLoS One 6, el7671 (2011 )).
• Excised eyes from these mice were treated with 5-cholesten-3b,25-diol (100 ⁇ M) or a saline control.
• 5-cholesten-3b,25-diol treatment significantly reduced the aggregation of cryAB and improved cryAB solubility.
• cryAB solubility and lens transparency were determined by measuring slit lens illumination in live animals and by gel permeation chromatography/light scattering from lens homogenates. This treatment significantly improved cryAB solubility, improved lens transparency and rescued cataract phenotypes in 10/15 treated R120G cryAB knock-in mice.
• 5-cholesten-3b,25-diol and more generally, a compound of formula I, is a promising therapeutic for the treatment of cataract. More broadly, these studies suggest that DSF-based HTS campaigns can be used to identify pharmacological chaperones for non-enzymes, such as cryAB.
• the data in Table 1 show a noticeable increase in median percent difference when the age is greater than 200 days.
• the invention is further embodied by the following items 1-27:

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